Lecture 4: Microbiology Pulse Batch - 2023 PDF

Pulse Batch - 2023 I C R O B I O L O GY M ُ ‫ﻠﻬﻢ‬ ِ ‫ﻣ‬-‫ﺗﻔﺎرﻳﻎ دﻓﻌﺔ ﻧﺒﺾ‬ LECTURE 4 DONE BY: Lana Yaseen...

Pulse Batch - 2023 I C R O B I O L O GY M ُ ‫ﻠﻬﻢ‬ ِ ‫ﻣ‬-‫ﺗﻔﺎرﻳﻎ دﻓﻌﺔ ﻧﺒﺾ‬ LECTURE 4 DONE BY: Lana Yaseen EDITED BY: Fadel Al-aswad  Microbial Diversity Dr. Waleed Al Momani, MLT, PhD Dr. Waleed Al Momani  Introduction Microbes can be divided into those that are truly cellular (bacteria, archaea, algae, protozoa, and fungi) and those that are acellular (viruses, viroids, and prions) The cellular microorganisms can be subdivided into those that are prokaryotic (bacteria and archaea) and those that are eukaryotic (algae, protozoa, and fungi). Acellular microorganisms are not considered by most scientists to be living organisms. viruses, viroids, and prions are more correctly referred to as acellular microbes or infectious particles. Dr. Waleed Al Momani  Acellular Microbes (Viruses) Viruses are extremely small. They are observed using electron microscopes. But due to developments in Microbiology tools, we’re now able to see some viruses with light microscopes. Viruses are not alive outside of live host cells. To replicate, viruses must invade live host cells. Viruses infect humans, animals, plants, fungi, protozoa, algae, and bacterial cells and they cause deadly diseases. Many human diseases are caused by viruses Oncogenic viruses or oncoviruses are viruses which cause specific types of cancer (tumors) including human cancers such as lymphomas, carcinomas, and some types of leukemia. Dr. Waleed Al Momani  Viruses have five specific properties: The vast majority of viruses possess either DNA or RNA, unlike living cells, which possess both. No virus has both DNA and RNA. Viruses can’t self replicate. It must enter a live cell and use the genetic machinery to be able to perform replication. And they depend on the ribosomes, enzymes, and metabolites (“building blocks”) of the host cell for protein and nucleic acid production. After they replicate, their number becomes huge. One of these things would happen: 1. the cell bursts. 2. The viruses replicate and protrude through the plasma membrane, in a process called “budding” (‫)التبرعم‬ Unlike cells, they do not divide by binary fission, mitosis, or meiosis. They lack the genes and enzymes necessary for energy production. Dr. Waleed Al Momani  Bacteriophages The viruses that infect bacteria cells are known as bacteriophages They will be integrated in the chromosome of the bacterial cell It will “infect” the bacterial cell with new genetic particles, so the bacteria will be pathogenic if the bacteriophage is pathogenic. An important example is the bacteria corynebacterium bacteria, which causes a dangerous disease called “diphtheria” (‫)مرض الخانوق‬ Normally, corynebacterium bacteria is non toxigenic. It becomes toxigenic when it’s infected with a virus. That’s when it causes diphtheria. Diphtheria is an upper respiratory tract disease. Viruses or bacteriophages are obligate intracellular pathogens, in that they must enter a cell to replicate. Bacteriophages can only attach to bacteria that possess surface molecules (receptors) that can be recognized by molecules on the phage surface. Dr. Waleed Al Momani  Categories of bacteriophages Bacteriophages can be categorized by: 1. the type of nucleic acid that they possess. DNA or RNA bacteriophages. 2. the events that occur after invasion of the bacterial cell, Classification based on events that occur some are virulent phages, whereas AFTER invasion of others are temperate phages. bacterial cell (This is the more important classification tool) When the viral genes integrate the bacterial chromosome, genes that are responsible for Virulent phages Temperate phages toxigenity become parts of the bacterial chromosome. So, when that chromosome duplicates and divides, it will still carry the toxigenic trait. So, the new two identical bacteria produced by binary fission will have the altered gene, which is very dangerous. Dr. Waleed Al Momani  Virulent phages: This type of bacteriophages always immediately initiates the lytic cycle, which results in the destruction of the cell. Steps of the lytic cycle: 1. Attachment >>> (Most bacteriophages are species-and strain-specific) (it means they don’t bind to random bacteria, they have specific targets that contain receptors) 2. Penetration >>> (The phage injects its DNA into the bacterial cell) 3. Biosynthesis >>> (the phage genes are expressed, resulting in the production (biosynthesis) of viral pieces) 4. Assembly >>> (using the genetic machinery of the host cell, the viral pieces are assembled to produce complete viral particles (virions). Viral DNA is packaged up into capsids). So, virions (complete viral particles) are the products of assembly of viral pieces. Caspids are protein costings enclosing the genetic material. 1. Release >>> (The host either cell bursts open and all of the new virions escape from the cell, or budding happens >>> resulting in enveloped particles.).  Temperate phages: This type of bacteriophages does not immediately initiate the lytic cycle. Their DNA can remain integrated into the host cell’s chromosome for generation after generation. They are involved in two of the four major ways in which bacteria acquire new genetic information >>> Bacteria can acquire new genetic information from other bacteria using one of these 4 ways: 1) Conjucation 2) Transformation 3) Transduction Viruses are 4) Lysogeny (lysogenic conversion) enveloped Temperate phages use both the Lysogenic conversion and the Transduction methods. Dr. Waleed Al Momani  Animal Viruses Viruses that infect humans and animals, not only animals. e.g. corona virus. One of the animals that are considered reservoirs for the corona virus are camels. In 2013, countries with an appropriate nature for camels experienced a high number of cases associated with corona virus. The mortality rate was more than 40% of the affected people (a very high mortality rate compared to the recent (2020) corona virus pandemic.) ‫على األغلب مو مهم‬ Like bacteriophages, animal viruses can only attach to and invade cells bearing appropriate surface receptors. But, bacteria and viruses can adapt. This theory is called frequency and microbial content. E.g. E. coli exists normally in the colon, but sometimes it’s found in the brain, forming brain abscess How did it reach the brain when the brain does not have a receptor for E coli? Because bacteria has the ability to ADAPT. If you’re frequently subjected to a bacteria or a virus that you don’t have a receptor for, it will get used to your body and it will find another way to attach to your cells. And that is abnormal. Animal viruses also escape from their host cells by either lysis of the cell or budding. Viruses that escape by budding become enveloped viruses. (In a double phospholipid layer [a part of the cell’s membrane] ) Dr. Waleed Al Momani  The mechanism of animal viruses Step Name of Step What Occurs During This Step 1 Attachment (adsorption) The virus attaches to a protein or polysaccharide molecule (receptor) on the surface of a host cell 2 Penetration The entire virus enters the host cell, in some cases because it was phagocytized by the cell 3 Uncoating The viral nucleic acid escapes from the capsid 4 Biosynthesis Viral genes are expressed, resulting in the production of pieces or parts of viruses (i.e., viral DNA and viral proteins) 5 Assembly The viral pieces or parts are assembled to create complete virions 6 Release The complete virions escape from the host cell by lysis or budding Dr. Waleed Al Momani  How do we diagnose a viral infection? - Culturing viruses is practically very hard. In every hospital, you’d find bacteriology, parasitology, micrology, but never virology. - Because virology is not culturable in clinical labs, because it needs specific environments, and there’s a danger in transmission. It’s either diagnosed by serology (‫)فحوصات األمصال‬, or under microscopes using smears to detect the cytopathic effects the virus has on a host cell. That cytopathic effect, is called inclusion bodies: - Inclusion bodies: remnants or collections of viruses that are often seen in infected cells and are used as a diagnostic tool to identify certain viral diseases. Inclusion bodies may be found in either the cytoplasm, or within the nucleus depending on the particular disease. Examples: 1. In rabies >>> the cytoplasmic inclusion bodies in nerve cells >>> Cytoplasmic they’re called Negri bodies (that’s inclusion bodies how you diagnose rabies under the microscope) Inclusion bodies Intranuclear 2. In Herpes and poliomyelitis >>> inclusion bodies intranuclear inclusion bodies Dr. Waleed Al Momani  Infections Latent infections Active infections Actual infections >> the symptoms are Hidden virus. showing on the patient.  Latent Virus Infections Latent viruses depend on the state of the immune system (weak, healthy) Herpes is one example of a latent infection. It causes ulcers (‫ )تقرحات‬on cheeks or on the tongue, or cold sores on the lips. Those ulcers open and close. Why? When the ulcers open, that means the immune system of the patient is weak and compromised. NOT because the virus is gone. (e.g. in stressful times, the immune system becomes weaker) When the ulcers close, that means your immune system was strong enough to close it. So, although the infected person is always harboring the virus in nerve cells, the cold sores come and go. Latent viral infections are usually limited by the defense systems of the human body Dr. Waleed Al Momani  Antiviral Agents Like we have antibiotics, antiviral drugs were also synthesized. These chemicals have been developed to interfere with virus-specific enzymes and virus production by either disrupting critical phases in viral cycles or inhibiting the synthesis of viral DNA, RNA, or proteins. But unfortunately, due the poor medical knowledge of most people, they would buy antibiotics whenever they have symptoms. But antibiotics do not work with viral infections. It might be suppressed (the activity of the virus might go down a little) but the virus will not be killed by antibiotics. It is very important for healthcare professionals to understand that antibiotics are not effective against viral infections. Misuse of antibiotics causes anti-microbial resistance. Dr. Waleed Al Momani  Oncogenic Viruses Viruses that cause cancer are called oncogenic viruses or oncoviruses Viruses were shown to be the cause of various types of cancers in rodents, frogs, and cats. It is known that some human cancers are caused by viruses An important example: Epstein–Barr virus. Epstein-Barr virus is very prevalent, meaning that it’s very widely spread and exists in all communities. So people with weakened immune systems could be susceptible for cancers like: Nasopharyngeal carcinoma, Burkitt lymphoma, and B-cell lymphoma. (All of them are caused by Epstein-Barr virus) Human papillomaviruses (HPV) If not treated quickly, it causes cancers of the cervix for females and other parts of the genital tract. Is transmitted sexually It has a vaccine Dr. Waleed Al Momani  Human Immunodeficiency Virus HIV is an enveloped, single-stranded RNA virus, which causes the AIDS disease. Since approx. 40 years, many attempts at controlling HIV have been made, but none of these attempts worked. Because the mutation rate of the virus is very high. It is a member of a genus of viruses called lentiviruses, in a family of viruses called Retroviridae (retroviruses) Mimivirus and Megavirus Mimivirus: Megavirus chilensis (Megavirus): Extremely large Discovered in 2010 off the coast of double-stranded DNA virus Chile can be observed using a standard compound Its genome is larger than that of some light microscope bacteria. It’s recovered from amebas. Megavirus was isolated in a French laboratory by co- cultivation with The virus was given the name Mimivirus because amebas. it “mimics” bacteria Its natural host is not known. Mimivirus may be the cause of some cases of human pneumonia Dr. Waleed Al Momani  Plant Viruses More than 1,000 different viruses cause plant diseases Plant viruses are usually transmitted via insects mites; nematodes (round worms); and contaminated tools. These diseases result in huge economic losses, estimated to be billions of dollars per year worldwide. But they do NOT affect humans. That’s why we eat virally infected food without getting ill. Viroids and Prions Viroids: small pieces of infectious RNA molecules cause a variety of plant diseases. consist of short, naked fragments of single- stranded RNA (about 300–400 nucleotides) Dr. Waleed Al Momani  Prions: Small infectious protein molecules that cause a variety of animal and human diseases. Especially neurological syndromes. ‫مهم‬ They infect both animals and humans. In animals, they cause fatal neurological diseases, such as scrapie in sheep and goats and bovine spongiform encephalopathy in cows (mad cow disease) In humans, they cause kuru, Creutzfeldt–Jakob (C–J) disease, Gerstmann–Strussler–Scheinker (GSS) disease, and fatal familial insomnia. Kuru, C–J, and GSS diseases involve loss of coordination and dementia. The most important one being dementia, which is a general mental deterioration, is characterized by disorientation and impaired memory, judgment, and intellect. (‫)مرض الخرف‬ The person who discovered prions won the 1997 Nobel Prize for Physiology or Medicine: Stanley B. Prusiner, the scientist who coined the term prion and studied the role of these proteinaceous infectious particles in disease. Dementia is not the same as Alzheimer’s disease, but they both have a relation to Prions and they’re both neurological diseases. Possible relationships between prions and Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis (ALS), some types of cancer, and type II diabetes. ‫الدكتور ذكر األمراض يلي باألصفر بهالفقرة‬ Dr. Waleed Al Momani  Bacteria Bacteria is the largest microbial group, and the one that causes the most human diseases. The Domain Bacteria contains 23 phyla, 32 classes, 5 subclasses, 77 orders, 14 suborders, 182 families, 871 genera, and 5,007 species. ‫ما ذكرهم الدكتور‬ Bacteria are divided into three phenotypic categories (i.e., categories based on their physical characteristics): 1. Gram-positive (thick cell walls) >>> 30+ layers of peptidoglycan >>> stains purple/blue 2. Gram-negative (thick cell walls) >>> 1-2 layers of peptidoglycan >>> stains red/pink 3. Those that lack a cell wall (cell wall-less) (cell wall deficient) (L-form) >>> like mycoplasma >>> appears colorless using gram stain. We use a specific type of stain called dienes stain with it. Some revert to their original shape when placed in favorable growth conditions, whereas others do not Cell Morphology Identification of bacteria depends on either the: Shape of bacteria (coccus, bacillus, and spiral shaped) The arrangement of cells together (singly or in pairs (diplococci), chains (streptococci), clusters (staphylococci), packets of four (tetrads), or packets of eight (octads)) The size, shape, and morphologic arrangement of various bacteria are easily observed using compound light microscope and the GRAM STAIN. The gram stain is a very old technique, used for more than 150 years now. It’s named after Mr. Gram (the scientist who invented it). Dr. Waleed Al Momani  There are types of stains: 1. Simple stains (one type of stain) >>> to see the shape and arrangement 2. Differential stains >>> to differentiate between two groups (e.g. the gram stain) 3. Structural stains >>> they stain specific structures within the bacterial cell (e.g. special flagellal stains, special capsule stains) In differential stains, we have a primary and contrary stain. For example: Gram-positive bacteria stains purple (primary stain) Gram-negative bacteria stains red (contrary stain) Bacteria vary greatly in size, usually ranging from 0.2 μm in diameter to 10.0-μm-long spiral- shaped bacteria, to even longer filamentous bacteria. A bacterium’s Gram reaction (Gram positive or Gram negative), basic cell shape, and morphological arrangement of the cells are very important clues to the organism’s identification. Gram-variable bacteria means they stain irregularly, and there's a mix of pink- and purple- colored bacteria= M. tuberculosis and M. leprae. A bacterial species having cells of Cocci: round different shapes is Bacilli: rod shaped said to be Spirilla: spiral shaped pleomorphic. Dr. Waleed Al Momani  Staining Procedures Most bacteria are colorless, transparent, and difficult to see ‫الصور ما علق عليها الدكتور‬ Fixation before staining serves three purposes: 1. It kills the organisms. 2. It preserves their morphology. 3. It anchors the smear to the slide. Dr. Waleed Al Momani  The acid-fast stain is of value in the diagnosis of tuberculosis. Acid-fast bacteria are red at the end of the acid-fast staining procedure. ‫الساليد ما انذكر ابدا‬ Dr. Waleed Al Momani  Motility Bacteria differ in their motile abilities, some are motile, some aren’t Motile bacteria = they swim Bacterial motility = caused by flagella or axial filaments Gliding motility = they have no flagella A flagellal stain = demonstrates the presence, number, and location of flagella on bacterial cells. To differentiate between the motile and the non motile types, we use 2 techniques: 1. Stabbing the bacteria into a tube of semisolid agar (it’s more precise) 2. Using the hanging drop technique (very simple, we look at the drop of water that has bacteria and see it under the microscope, if it moves or not) Dr. Waleed Al Momani  We stab a needle that has bacteria in a semi solid agar (50% agar) The stabbing has to be in a straight line Then we incremate it (the bacteria) Then, it appears whether the bacteria is motile or non-motile. It diffused on the sides of the needle It grew on the line (it moved) same needle line Dr. Waleed Al Momani